Dyes in general, and fluorescent dyes in particular, are often used to directly stain or label a sample so that the sample can be identified or quantitated. For instance, such dyes may be added as part of an assay for a biological target analyte, as a detectable tracer element in a biological or non-biological fluid, or for such purposes as photodynamic therapy of tumors, in which a dyed sample is irradiated to selectively destroy tumor cells and tissues, or to photoablate arterial plaque or cells. Furthermore, fluorescent dyes can often be chemically modified so that they can be covalently attached to a variety of materials to give them desirable fluorescent characteristics.
Fluorescent dyes with longer wavelength absorption and emission are particularly useful in conjunction with materials of biological origin such as blood, urine, fecal matter, cells and tissues, where background or inherent fluorescence or absorption often interferes with detection of the added fluorescent dye. Furthermore, biological specimens often have decreasing levels of both absorption and fluorescence emission as the illumination energy approaches the infrared. In addition, numerous biological and nonbiological applications of long wavelength dyes exist, including use as laser dyes, or in electronics as optical memory elements using relatively low cost illumination sources such as laser diodes. Consequently, dyes that possess these spectral properties have potential utility in biological and non-biological applications.
A variety of dipyrrometheneboron difluoride dyes (4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes) have previously been described, by these inventors, and others, in which substituents are attached by a single covalent bond, including U.S. Pat. Nos. 4,774,339 to Haugland et al. and U.S. Pat. No. 5,274,113 to Kang et al. (reactive substituents), U.S. Pat. No. 5,248,782 to Haugland et al. (heteroaryl substituents), U.S. Pat. No. 5,178,288 to Kang et al. (ethenyl substituents), and Ser. No. 07/654,881 by Kang et al., filed 2/13/91 (fatty acid substituents). All the above issued and pending patents describe fluorescent dyes and conjugates of fluorescent dyes based on the dipyrrometheneboron difluoride core structure shown below: ##STR2##
The use of derivatives of dipyrrometheneboron difluoride (particularly symmetrical alkyl derivatives and alkyl derivatives that are also sulfonated) as laser dyes is described in U.S. Pat. No. 4,916,711 to Boyer, et al.
None of the above references described dipyrrometheneboron difluoride dyes that incorporate fused aromatic rings, as found in the compounds of the present invention that are based on the 3,4:3',4'-dibenzopyrrometheneboron difluoride core structure below: ##STR3##
The synthetic route to the dibenzopyrrometheneboron difluoride dyes (described in detail below) proceeds through the condensation of 2-acylacetophenones. This synthetic route is very different from that used for previously described dipyrrometheneboron difluoride fluorophores. Furthermore, the dyes of the present invention cannot be prepared using the previous method, due to the instability of the isoindole nucleus.
Aryl-substituted derivatives of the key intermediate in the synthesis of some of the instant dyes, dibenzopyrromethenes, are isolable compounds that were originally described by Maekawa et al. (CHEM. BER. 101, 847 (1968)). This reference does not teach or recommend the conversion of the dibenzopyrromethene intermediate to the subject dyes, and does not indicate that such dyes would possess any of the desirable properties possessed by the dyes of the present invention.
The dyes of the present invention typically possess longer wavelength excitation and emission bands than previously described dipyrrometheneboron difluoride dyes. In addition, the spectral properties of the dyes are substantially insensitive to the chemical environment of the dye, even when compared to the previously described dipyrrometheneboron difluoride dyes. Practically this means that the absorbance and emission of the dyes differ only slightly when measured in different solvents. The subject dyes are stable to photobleaching, remaining intensely fluorescent even when constantly illuminated. Finally, selected dyes of the present invention possess exceptionally narrow excitation bands coupled with very high extinction coefficients. The combination of these favorable properties make dibenzopyrrometheneboron difluoride dyes exceptionally useful fluorophores for a variety of applications.